title: Identification and real-time expression analysis of selected Toxoplasma gondii in-vivo induced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients.
abstract: BACKGROUND
  Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
  
  
  METHODS
  The pooled sera was first pre-absorbed against three different preparations of antigens from in-vitro-grown cells of each T. gondii and E. coli XL1-Blue MRF', subsequently it was used to screen T. gondii cDNA phage expression library. Positive clones from each group were subjected to quantitative real-time PCR expression analysis on mRNA of in-vivo and in-vitro grown parasites.
  
  
  RESULTS
  A total of 29 reactive clones from each IgM and IgG immunoscreenings were found to have high homology to T. gondii genes. Quantitative real-time PCR expression analysis showed that 20 IgM-detected genes and 11 IgG-detected genes were up-regulated in-vivo relative to their expression levels in-vitro. These included genes encoding micronemes, sterol-regulatory element binding protein site, SRS34A, MIC2-associated protein M2AP, nucleoredoxin, protein phosphatase 2C and several hypothetical proteins. A hypothetical protein (GenBank accession no. 7899266) detected by IgG had the highest in-vivo over in-vitro fold change of 499.86; while another up-regulated hypothetical protein (GenBank accession no. 7898829) recognized by IgM showed high sensitivity (90%) and moderate specificity (70%) in detecting T. gondii antibodies when tested with 20 individual serum samples.
  
  
  CONCLUSIONS
  The highly up-regulated genes and the corresponding proteins, in particular the hypothetical proteins, may be useful in further studies on understanding the disease pathogenesis and as potential vaccine candidates.
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang 11800, Malaysia.
authors.forename: Atefeh
authors.initials: A
authors.lastname: Amerizadeh
authors.forename: Boon Yin
authors.initials: BY
authors.lastname: Khoo
authors.forename: Ai Ying
authors.initials: AY
authors.lastname: Teh
authors.forename: Majid
authors.initials: M
authors.lastname: Golkar
authors.forename: Izzati Zahidah
authors.initials: IZ
authors.lastname: Abdul Karim
authors.forename: Sabariah
authors.initials: S
authors.lastname: Osman
authors.forename: Muhammad Hafiznur
authors.initials: MH
authors.lastname: Yunus
authors.forename: Rahmah
authors.initials: R
authors.lastname: Noordin
mesh_terms: D000327:Adsorption; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology* / Q000378:metabolism; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D001709:Biotechnology / Q000379:methods*; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D007075:Immunoglobulin M / Q000097:blood / Q000276:immunology*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics / Q000276:immunology*; D014123:Toxoplasmosis / Q000097:blood / Q000276:immunology* / Q000382:microbiology; D014709:Vero Cells
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D018076:DNA, Complementary; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins